Systems and methods for collecting tear film and measuring tear film osmolarity

ABSTRACT

A sample receiving chip comprising a substrate that receives an aliquot volume of a sample fluid and a sample region of the substrate, sized such that the volume of the sample fluid is sufficient to operatively cover a portion of the sample region. The energy imparted into the sample fluid is transduced by the sample region to produce an output signal that indicates energy properties of the sample fluid. The sample receiving chip also includes a channel formed in the substrate, the channel configured to collect the aliquot volume of a sample fluid and transfer the aliquot volume of sample fluid to the sample region.

RELATED APPLICATION INFORMATION

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application Ser. No. 60/869,543, entitled “Systemsand Methods for Collecting Tear Film and Measuring Tear FilmOsmolarity,” by Eric Donsky et al., filed Dec. 11, 2006. ThisApplication also claims the benefit as a Continuation-In-Part under 35U.S.C. 120 of co-pending U.S. patent application Ser. No. 10/400,617entitled “Tear Film Osmometry”, by Benjamin D. Sullivan, filed Mar. 25,2003. Both of the above applications are incorporated herein byreference as if set forth in full.

BACKGROUND

1. Technical Field

The embodiments described herein relate generally to measuring theosmotic pressure of fluids and, more particularly, to measuring theosmolarity of tear film.

2. Related Art

Tears fulfill an essential role in maintaining ocular surface integrity,protecting against microbial challenge, and preserving visual acuity.These functions, in turn, are critically dependent upon the compositionand stability of the tear film structure, which includes an underlyingmucin foundation, a middle aqueous component, and an overlying lipidlayer. Disruption, deficiency, or absence of the tear film can severelyimpact the eye. If unmanaged with artificial tear substitutes or tearfilm conservation therapy, these disorders can lead to intractabledesiccation of the corneal epithelium, ulceration and perforation of thecornea, an increased incidence of infectious disease, and ultimatelypronounced visual impairment and blindness.

Keratoconjunctivitis sicca (KCS), or “dry eye”, is a condition in whichone or more of the tear film structure components listed above ispresent in insufficient volume or is otherwise out of balance with theother components. It is known that the fluid tonicity or osmolarity oftears increases in patients with KCS. KCS is associated with conditionsthat affect the general health of the body, such as Sjogren's syndrome,aging, and androgen deficiency. Therefore, osmolarity of a tear film canbe a sensitive and specific indicator for the diagnosis of KCS and otherconditions.

The osmolarity of a sample fluid, e.g., a tear, can be determined by anex vivo technique called “freezing point depression,” in which solutesor ions in a solvent, i.e. water, cause a lowering of the fluid freezingpoint from what it would be without the ions. In freezing pointdepression analysis, the freezing point of the ionized sample fluid isfound by detecting the temperature at which a quantity of the sample,typically on the order of about several milliliters, first begins tofreeze in a container, e.g., a tube. To measure the freezing point, avolume of the sample fluid is collected into a container, such as atube. Next, a temperature probe is immersed in the sample fluid, and thecontainer is brought into contact with a freezing bath or Peltiercooling device. The sample is continuously stirred so as to achieve asupercooled liquid state below its freezing point. Upon mechanicalinduction, the sample solidifies, rising to its freezing point due tothe thermodynamic heat of fusion. The deviation from the sample freezingpoint from 0° C. is proportional to the solute level in the samplefluid. This type of measuring device is sometimes referred to as afreezing point depression osmometer.

Presently, freezing point depression measurements are made ex vivo byremoving tear samples from the eye using a micropipette or capillarytube, expelling the tear samples into a cup, and measuring thedepression of the freezing point that results from heightenedosmolarity. However, these ex vivo measurements are often plagued bymany difficulties. For example, to perform freezing point depressionanalysis of the tear sample, a relatively large volume must becollected, typically on the order of 1-5 microliters (μL) of tear film.Because no more than about 10 to 100 nanoliters (nL) of tear sample canbe obtained at any one time from a KCS patient, the collection ofsufficient amounts of fluid for conventional ex vivo techniques requiresa physician to induce reflex tearing in the patient. Reflex tearing iscaused by a sharp or prolonged irritation to the ocular surface, akin towhen a large piece of dirt becomes lodged in one's eye. Reflex tears aremore dilute, i.e. have fewer solute ions than the tears that arenormally found on the eye. Any dilution of the tear film invalidates thediagnostic ability of an osmolarity test for dry eye, and therefore makecurrently available ex vivo methods prohibitive in a clinical setting.

A similar ex vivo technique is vapor pressure osmometry, where a small,circular piece of filter paper is lodged underneath a patient's eyeliduntil sufficient fluid is absorbed. The filter paper disc is placed intoa sealed chamber, whereupon a cooled temperature sensor measures thecondensation of vapor on its surface. Eventually the temperature israised to the dew point of the sample. The reduction in dew pointproportional to water is then converted into osmolarity. Because of theinduction of reflex tearing and the large volume requirements forexisting vapor pressure osmometers, they are currently impractical fordetermination of dry eye.

The Clifton Nanoliter Osmometer, available from Clifton TechnicalPhysics of Hartford, N.Y., USA, is a freezing point depression osmometerand has been used extensively in laboratory settings to quantify thesolute concentrations of KCS patients, but the machine requires asignificant amount of training to operate. It generally requireshour-long calibrations and a skilled technician in order to generateacceptable data. The Clifton Nanoliter Osmometer is also bulky andrelatively expensive. These characteristics invalidate its use as aclinical osmometer.

In contrast to ex vivo techniques that measure osmolarity of tearsamples removed from the ocular surface, an in vivo technique thatattempted to measure osmolarity directly on the ocular surface used aflexible pair of electrodes that were placed directly underneath theeyelid of the patient. The electrodes were then plugged into an LCRmeter to determine the conductivity of the fluid surrounding them. Whileit has long been known that conductivity is directly related to theionic concentration, and hence osmolarity of solutions, placing thesensor under the eyelid for half a minute likely induced reflex tearing.Moreover, the electrodes are difficult to manufacture and pose increasedhealth risks to the patient as compared to simply collecting tears witha capillary. Moreover, many DES patients exhibit a discontinuous tearlake, such that the curvature of the discontinuity would substantiallyalter the measured conductivity using an exposed probe, increasinguser-to-user variability.

It should be apparent from the discussion above that current osmolaritymeasurement techniques are unavailable in a clinical setting and can'tattain the volumes necessary for dry eye patients. Thus, there is a needfor an improved, clinically feasible, nanoliter-scale osmolaritymeasurement.

SUMMARY OF THE INVENTION

Osmolarity measurement of a sample fluid, such as a tear film, isachieved by collecting an aliquot volume of the sample fluid and causingthe aliquot volume of the sample fluid to come in contact with a samplereceiving chip comprising a substrate that receives an aliquot volume ofa sample fluid, a sample region of the substrate, sized such that thevolume of the sample fluid is sufficient to operatively cover a portionof the sample region, whereupon energy properties of the sample fluidcan be detected from the sample region to produce a sample fluidreading, wherein the sample fluid reading indicates osmolarity of thesample fluid.

In one aspect, an osmolarity measurement of the sample fluid can beobtained from the detected energy properties of the sample volume.

In another aspect, the aliquot-sized volume of sample fluid can bequickly and easily obtained, even from dry eye patients using a samplereceiving substrate formed within a microchip. In one embodiment, thesample receiving substrate is comprised of a specially constructedmicrofluidic channel that wicks the tear directly across the sampleregion. This embodiment eliminates the need to transfer the fluid,limits the amount of evaporation, and helps reduce user-to-uservariability.

An aliquot volume can comprise, for example, a volume of no more than 20microliters (μL), but can be as little as 1 nL. An osmolarity sensorsystem can receive the microchip and sample volume, and can detectenergy from the sample volume to display an accurate osmolaritymeasurement. In this way, a reliable osmolarity measurement can beobtained with minimum inconvenience and discomfort to a patient, withoutrequiring a great deal of skill to obtain the measurement, and with ahigh degree of repeatability and accuracy.

Other features and advantages of the present invention should beapparent from the following description of the preferred embodiment,which illustrates, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an aliquot-sized sample receiving chip for measuringthe osmolarity of a sample fluid in accordance with one embodiment.

FIG. 2 illustrates an alternative embodiment of a sample receiving chipthat includes a circuit region with an array of electrodes imprintedwith photolithography techniques.

FIG. 3 illustrates another alternative embodiment of the FIG. 1 chip,wherein a circuit region includes printed electrodes arranged in aplurality of concentric circles.

FIG. 4 is a top view of the chip shown in FIG. 2.

FIG. 5 is a top view of the chip shown in FIG. 3.

FIG. 6 is a block diagram of an osmolarity measurement system configuredin accordance with one embodiment.

FIG. 7 is a perspective view of a tear film osmolarity measurementsystem constructed in accordance with one embodiment.

FIG. 8 is a side section of the sample receiving chip showing theopening in the exterior packaging in accordance with one embodiment.

FIG. 9 is a calibration curve relating the sodium content of the samplefluid with electrical conductivity.

FIG. 10 illustrates a hinged base unit of the osmometer that utilizesthe sample receiving chips described in FIGS. 1-5.

FIG. 11 illustrates a probe card configuration for the sample receivingchip and processing unit.

FIG. 12 illustrates an optical osmolarity measurement system constructedin accordance with one embodiment.

FIG. 13 is a flowchart describing an exemplary osmolarity measurementtechnique in accordance with one embodiment.

FIG. 14 is a side section of the sample receiving chip showing theopening in the exterior packaging in accordance with another embodiment.

FIG. 15 illustrated an example collection device that can hold thereceiving chip of FIG. 14.

FIG. 16 is a cross sectional view of a channel that can be formed in thereceiving chip of FIG. 14.

FIG. 17A illustrates a microfluidic collection device in accordance withone embodiment.

FIG. 17B illustrates a more detailed view of the device of FIG. 17A.

FIG. 17C illustrates an exploded view of a portion of the device of FIG.17B.

FIG. 18 is a diagram illustrating another example embodiment of amicrofluidic collection device.

FIG. 19 is a graph illustrating the change in osmolarity over timewithin a receiving substrate.

FIG. 20 is a graph illustrating the change in osmolarity over time forthree different tear collection interfaces.

DETAILED DESCRIPTION

Exemplary embodiments are described for measuring the osmolarity of analiquot volume of a sample fluid (e.g., tear film, sweat, blood, orother fluids). The exemplary embodiments are configured to be relativelyfast, non-invasive, inexpensive, and easy to use, with minimal risk ofinjury to the patient. Accurate measurements can be provided with aslittle as nanoliter volumes of a sample fluid. For example, a measuringdevice configured in accordance with the embodiments described hereincan enable osmolarity measurement with no more than 20 μL of samplefluid, and typically much smaller volumes can be successfully measured.In one embodiment described further below, osmolarity measurementaccuracy is not compromised by variations in the volume of sample fluidcollected, so that osmolarity measurement is substantially independentof collected volume. The sample fluid can include tear film, sweat,blood, urine or other bodily fluids. It should be noted, however, thatsample fluid can comprise other fluids, such as milk or other beverages.

FIG. 1 illustrates an exemplary embodiment of an osmolarity chip 100that can be used to measure the osmolarity of a sample fluid 102, suchas a tear film sample. In the example of FIG. 1, the chip 100 includes asubstrate 104 with a sample region having sensor electrodes 108 and 109and circuit connections 110 imprinted on the substrate. The electrodes108 and 109 and circuit connections 110 are preferably printed usingwell-known photolithographic techniques. For example, current techniquesenable the electrodes 108 and 109 to have a diameter in the range ofapproximately one (1) to eighty (80) microns, and spaced apartsufficiently so that no conductive path exists in the absence of samplefluid. Currently available techniques, however, can provide electrodesof less than one micron in diameter, and these are sufficient for a chipconstructed in accordance with the embodiments described herein.

The amount of sample fluid needed for measurement is no more than isnecessary to extend from one electrode to the other, thereby providingan operative conductive path. The photolithographic scale of the chip100 permits the measurement to be made for aliquot-sized samples in amicro- or nano-scale level. For example, reliable osmolarity measurementcan be obtained with a sample volume of less than 20 μL of tear film. Atypical sample volume can be less than one hundred nanoliters (100 nL).It is expected that it will be relatively easy to collect 10 nL of atear film sample even from patients suffering from dry eye.

The chip 100 can be configured to transfer energy to the sample fluid102 and enable detection of the sample fluid energy properties. In thisregard, a current source can be applied across the electrodes 108 and109 through the connections 110. The osmolarity of the sample fluid canbe measured by sensing the energy transfer properties of the samplefluid 102. The energy transfer properties can include, for example,electrical conductivity, such that the impedance of the sample fluid ismeasured, given a particular amount of electrical power, e.g., current,that is transferred into the sample through the connections 110 and theelectrodes 108 and 109.

If conductivity of the sample fluid is to be measured, then preferably asinusoidal signal on the order of ten volts at approximately 10 kHz isapplied. The real and imaginary parts of the complex impedance of thecircuit path from one electrode 108 through the sample fluid 102 to theother electrode 109 are measured. At the frequencies of interest, it islikely that the majority of the electrical signal will be in the realhalf of the complex plane, which reduces to the conductivity of thesample fluid. This electrical signal (hereafter referred to asconductivity) can be directly related to the ion concentration of thesample fluid 102, and the osmolarity can be determined. Moreover, if theion concentration of the sample fluid 102 changes, the electricalconductivity and the osmolarity of the fluid will change in acorresponding manner. Therefore, the osmolarity can be reliablyobtained. In addition, because the impedance value does not depend onthe volume of the sample fluid 102, the osmolarity measurement can bemade substantially independent of the sample volume.

As an alternative to the input signal described above, more complexsignals can be applied to the sample fluid the response of which willcontribute to a more thorough estimate of osmolarity. For example,calibration can be achieved by measuring impedances over a range offrequencies. These impedances can be either simultaneously, via combinedwaveform input and Fourier decomposition, or sequentially measured. Thefrequency versus impedance data will provide information about thesample and the relative performance of the sample fluid measurementcircuit.

FIG. 2 illustrates an alternative embodiment of a sample receiving chip200 that can be configured to measure osmolarity of a sample fluid 202,wherein the chip comprises a substrate layer 204 with a sample region206 comprising an imprinted circuit that includes an array of electrodes208. In the illustrated embodiment of FIG. 2, the sample region 206 hasa 5-by-5 array of electrodes that are imprinted with photolithographictechniques, with each electrode 208 having a connection 210 to one sideof the substrate 204. Not all of the electrodes 208 in FIG. 2 are shownwith a connection, for simplicity of illustration. The electrode canprovide measurements to a separate processing unit, described furtherbelow.

The electrode array of FIG. 2 can provide a means to measure the size ofthe tear droplet 202 by detecting the extent of conducting electrodes208 to thereby determine the extent of the droplet. In particular,processing circuitry can determine the number of electrodes that areconducting, and therefore the number of adjacent electrodes that arecovered by the droplet 202 can be determined. The planar area of thesubstrate that is covered by the sample fluid can thereby be determined.With a known nominal surface tension of the sample fluid, the height ofthe sample fluid volume over the planar area can also be reliablyestimated, and therefore the volume of the droplet 202 can bedetermined.

FIG. 3 illustrates another alternative embodiment of a sample receivingchip 300 on which a sample fluid 302 is deposited. The chip comprises asubstrate layer 304, wherein a sample region 306 is provided withelectrodes 308 that are configured in a plurality of concentric circles.In a manner similar to the square array of FIG. 2, the circulararrangement of the FIG. 3 electrodes 308 can also provide an estimate ofthe size of the sample fluid volume 302 because the droplet typicallycovers a circular or oval area of the sample region 302. Processingcircuitry can be configured to detect the largest (outermost) circle ofelectrodes that are conducting, and thereby determine a planar area ofcoverage by the fluid sample. As before, the determined planar area canprovide a volume estimate, in conjunction with a known surface tensionand corresponding volume height of the sample fluid 302. In the exampleof FIG. 3, the electrodes 308 can be printed using well-knownphotolithography techniques that currently permit electrodes to have adiameter in the range of one (1) to eighty (80) microns. This allows thesub-microliter droplet to substantially cover the electrodes. Theelectrodes can be printed over an area sized to receive the samplefluid, generally covering 1 mm² to 1 cm².

The electrodes and connections shown in FIG. 1, FIG. 2, and FIG. 3 canbe imprinted on the respective substrate layers as electrodes withcontact pads, using photolithographic techniques. For example, theelectrodes can be formed with different conductive metalization such asaluminum, platinum, titanium, titanium-tungsten, and other similarmaterial. In one embodiment, the electrodes can be formed with adielectric rim to protect field densities at the edges of theelectrodes. This can reduce an otherwise unstable electric field at therim of the electrode.

Top views of the exemplary embodiments of the chips 200 and 300 areillustrated in FIG. 4 and FIG. 5, respectively. The embodiments show thedetailed layout of the electrodes and the connections, and illustratehow each electrode can be electrically connected for measuring theelectrical properties of a sample droplet. As mentioned above, thelayout of the electrodes and the connections can be imprinted on thesubstrate 100, 200, 300 using well-known photolithographic techniques.

FIG. 6 is a block diagram of an osmometry system 600, configured inaccordance with an embodiment, showing how information is determined andused in a process that determines osmolarity of a sample fluid. Theosmometry system 600 can include a measurement device 604 and aprocessing device 606. The measurement device can receive a volume ofsample fluid from a collection device 608. The collection device 608 cancomprise, for example, a micropipette or capillary tube. The collectiondevice 608 can be configured to collect a sample tear film of a patient,such as by using negative pressure from a fixed-volume micropipette orcharge attraction from a capillary tube to draw a small tear volume fromthe vicinity of the ocular surface of a patient.

The measurement device 604 can comprise a system that transfers energyto the fluid in the sample region and detects the imparted energy. Forexample, the measurement device 604 can comprise circuitry that provideselectrical energy in a specified waveform, such as from a functiongenerator, to the electrical path comprising two electrodes bridged bythe sample fluid. The processing device 606 can be configured to detectthe energy imparted to the sample fluid and determine osmolarity. Theprocessing device can comprise, for example, a system including an RLCmultimeter that produces data relating to the reactance of the fluidthat forms the conductive path between two electrodes, and including aprocessor that determines osmolarity through a table look-up scheme. Ifdesired, the processing device can be housed in a base unit thatreceives one of the chips described above.

As mentioned above, a sample sufficient to provide an osmolaritymeasurement can contain less than 20 microliters (μL) of fluid. Atypical sample of tear film can be collected by a fluid collector suchas a capillary tube, which often contains less than one microliter oftear film. Medical professionals will be familiar with the use ofmicropipettes and capillary tubes, and will be able to easily collectthe small sample volumes described herein, even in the case of dry eyesufferers.

The collected sample fluid can be expelled from the collection device608 to the measurement device 604. The collection device can bepositioned above the sample region of the chip substrate either manuallyby a medical professional or by being mechanically guided over thesample region. In one embodiment, for example, the collection device,e.g., a capillary tube, is mechanically guided into position with aninjection-molded plastic hole in a base unit, or is fitted to a set ofclamps with precision screws, e.g., a micromanipulator with needles formicrochip interfaces. In another embodiment, the guide is acomputer-guided feedback control circuitry that holds the capillary tubeand automatically lowers it into the proper position.

The electrodes and connections of the chips measure energy properties ofthe sample fluid, such as conductivity, and enable the measuredproperties to be received by the processing device 606. The measuredenergy properties of the sample fluid include electrical conductivityand can also include other parameters, such as both parts of the compleximpedance of the sample, the variance of the noise in the output signal,and the measurement drift due to resistive heating of the sample fluid.The measured energy properties are processed in the processing device606 to provide the osmolarity of the sample. In one embodiment, theprocessing device 606 comprises a base unit that can accept a chip andcan provide electrical connection between the chip and the processingdevice 606. In another embodiment, the base unit can include a displayunit for displaying osmolarity values. It should be noted that theprocessing device 606 and, in particular, the base unit can be ahand-held unit.

FIG. 7 is a perspective view of a tear film osmolarity measuring system700 constructed in accordance with one embodiment. In the illustratedembodiment of FIG. 7, the exemplary system 700 includes a measuring unit701 that comprises a chip, such as one of the chips described above, anda connector or socket base 710, which provides the appropriatemeasurement output. The system 700 can be configured to determineosmolarity by measuring electrical conductivity of the sample fluid.Therefore, the measurement chip 701 can comprise a integrated circuit(IC) chip with a substrate having a construction similar to that of thechips described above in connection with FIG. 1 through FIG. 5. Thus,the chip 701 can include a substrate layer with a sample region that isdefined by at least two electrodes printed onto the substrate layer. Itwill be understood that such details are of a scale too small to bevisible in FIG. 7, but see FIG. 1 through FIG. 5, examples of thesedetails. The substrate and sample region can be encased within an inertpackage, in a manner that will be known to those skilled in the art. Inparticular, the chip 701 can be fabricated using conventionalsemiconductor fabrication techniques into an IC package 707 thatincludes electrical connection legs 708 that permit electrical signalsto be received by the chip 701 and output to be communicated outside ofthe chip. The packaging 707 can provide a casing that makes handling ofthe chip more convenient and helps reduce evaporation of the samplefluid.

FIG. 8 shows that the measurement chip 701 can be fabricated with anexterior opening hole 720 into which the sample fluid 702 can beinserted. Thus, the hole 720 can be formed in the semiconductorpackaging 707 to provide a path through the chip exterior to thesubstrate 804 and the sample region 806. The collection device, such asa micropipette or capillary tube, 808 can be positioned into the hole720 such that the sample fluid 702 is expelled from the collectiondevice directly onto the sample region 806 of the substrate 804. Thehole 720 can be sized to receive the tip of the collection device. Thehole 720 forms an opening or funnel that leads from the exterior of thechip onto the sample region 806 of the substrate 804. In this way, thesample fluid 702 can be expelled from the collection device 808 and canbe deposited directly on the sample region 806 of the substrate 804. Thesample region can be sized to receive the volume of sample fluid fromthe collection device. In FIG. 8, for example, the electrodes can form asample region 806 that is generally in a range of approximately 1 mm² to1 cm² in area.

Returning to FIG. 7, the chip 701 can include processing circuitry 704that comprises, for example, a function generator that generates asignal of a desired waveform, which can be applied to the sample regionelectrodes of the chip, and a voltage measuring device to measure theroot-mean-square (RMS) voltage value that is read from the chipelectrodes. The function generator can be configured to produce highfrequency alternating current (AC) to avoid undesirable direct current(DC) effects for the measurement process. The voltage measuring devicecan incorporate the functionality of an RLC measuring device. Thus, thechip 701 can incorporate the measurement circuitry as well as the sampleregion electrodes. The processing circuitry can include a centralprocessing unit (CPU) and associated memory that can store programminginstructions (such as firmware) and also can store data. In this way, asingle chip can include the electrodes and associated connections forthe sample region, and on a separate region of the chip, can alsoinclude the measurement circuitry. This configuration can minimize theassociated stray resistances of the circuit structures.

As noted above, the processing circuitry 704 can be configured to applya signal waveform to the sample region electrodes. The processingcircuitry can also receive the energy property signals from theelectrodes and determine the osmolarity value of the sample fluid. Forexample, the processing unit can receive electrical conductivity valuesfrom a set of electrode pairs. Those skilled in the art will be familiarwith techniques and circuitry for determining the conductivity of asample fluid that forms a conducting path between two or moreelectrodes.

In the example of FIG. 7, the processing unit 704 can be configured toproduce signal waveforms at a single frequency, such as 100 kHz and 10Volts peak-to-peak. The processing circuitry 704 can then determine theosmolarity value from the sodium content correlated to the electricalconductivity using a calibration curve, such as the curve shown in FIG.9. In this case, the calibration curve can be constructed as a transferfunction between the electrical conductivity (voltage) and theosmolarity value, i.e., the sodium content. It should be noted, however,that other calibration curves can also be constructed to providetransfer functions between other energy properties and the osmolarityvalue. For example, the variance, autocorrelation and drift of thesignal can be included in an osmolarity calculation. If desired, theosmolarity value can also be built upon multi-variable correlationcoefficient charts or neural network interpretation so that theosmolarity value can be optimized with an arbitrarily large set ofmeasured variables.

In an alternative to the embodiment shown in FIG. 7, the processing unit704 can be configured to produce signal waveforms of a predeterminedfrequency sweep, such as 1 kHz to 100 kHz in 1 kHz increments, and storethe conductivity and variance values received from the set of electrodepairs at each frequency. The output signal versus frequency curve canthen be used to provide higher order information about the sample, whichcan be used with the aforementioned transfer functions to produce anideal osmolarity reading.

As shown in FIG. 7, the base socket connector 710 can receive the pins708 of the chip 701 into corresponding sockets 711. The connector 710,for example, can supply the requisite electrical power to the processingcircuitry 704 and electrodes of the chip. Thus, the chip 701 can includethe sample region electrodes and the signal generator and processingcircuitry necessary for determining osmolarity, and the outputcomprising the osmolarity value can be communicated off the chip via thepins 708 through the connector 710 and to a display readout.

If desired, the base connector socket 710 can include a Peltier layer712 located beneath the sockets that receive the pins 708 of the chip701. Those skilled in the art will understand that a Peltier layercomprises an electrical/ceramic junction such that properly appliedcurrent can cool or heat the Peltier layer. In this way, the sample chip701 can be heated or cooled, thereby further controlling evaporation ofthe sample fluid. It should be apparent that evaporation of the samplefluid should be carefully controlled, to ensure accurate osmolarityvalues obtained from the sample fluid.

FIG. 10 shows an alternative embodiment of an osmometer in which thechip does not include an on-chip processing unit such as describedabove, but rather includes limited circuitry comprising primarily thesample region electrodes and interconnections. That is, the processingunit is separately located from the chip and can be provided in, e.g.,the base unit.

FIG. 10 shows in detail an osmometer 1000 that includes a base unit1004, which houses the base connector 710, and a hinged cover 1006 thatcloses over the base connector 710 and a received measurement chip 701.Thus, after the sample fluid has been dispensed on the chip, the chipcan be inserted into the socket connector 710 of the base unit 1004 andthe hinged cover 1006 can be closed over the chip to reduce the rate ofevaporation of the sample fluid.

It should be noted that the problem with relatively fast evaporation ofthe sample fluid can generally be handled in one of two ways. One way isto measure the sample fluid voltage quickly and as soon possible afterthe droplet is placed on the sample region of the chip. Another way isto enable the measuring unit to measure the rate of evaporation alongwith the corresponding changes in conductivity values. The processingunit can then post-process the output to estimate the osmolarity value.The processing can be performed in the hardware and/or in softwarestored in the hardware. Thus, the processing unit can incorporatedifferent processing techniques such as using neural networks to collectand learn about characteristics of the fluid samples being measured forosmolarity, as well as temperature variations, volume changes, and otherrelated parameters so that the system can be trained in accordance withneural network techniques to make faster and more accurate osmolaritymeasurements.

FIG. 11 shows another alternative construction, in which the osmolaritysystem uses a sample receiving chip 1102 that does not include ICpackaging such as shown in FIG. 7. Rather, the FIG. 11 measurement chip1102 is configured as a chip with an exposed sample region comprisingthe electrodes and associated connections, but the processing circuitryis located in the base unit for measuring the energy properties of thesample fluid. In this alternative construction, a connector similar tothe connector socket 710 can allow transmission of measured energyproperties to the processing unit in the base unit. Those skilled in theart will understand that such a configuration is commonly referred to aprobe card structure.

FIG. 11 shows a probe card base unit 1100 that can receive a sample chipprobe card 1102 that comprises a substrate 1104 with a sample region1106 on which are formed electrodes 1108 that can be wire bonded to edgeconnectors 1110 of the probe card. When the hinged lid 1112 of the baseunit is closed down over the probe card, connecting tines 1114 on theunderside of the lid can be configured to come into mating contact withthe edge connectors 1110. In this way, the electrodes of the sampleregion 1106 can be coupled to the processing circuitry and measurementcan take place. The processing circuitry of the probe card embodiment ofFIG. 11 can, e.g., be configured in either of the configurationsdescribed above. That is, the processing to apply current to theelectrodes and to detect energy properties of the sample fluid anddetermine osmolarity can be located on-chip, on the substrate of theprobe card 1102, or the processing circuitry can be located off-chip, inthe base unit 1100.

In all the alternative embodiments described above, the osmometer a newmeasurement chip can be placed into the base unit while the hinged topis open. Upon placement into the base unit, the chip can be powered upand begin monitoring its environment. Recording output signals from thechip at a rate of, for example, 1 kHz, should fully capture the behaviorof the system. Placing a sample onto any portion of the electrode arrayshould generate high signal-to-noise increase in conductivity betweenany pair of electrodes covered by the sample fluid. The processing unitcan then recognize the change in conductivity as being directly relatedto the addition of sample fluid, and can begin conversion of electronicsignals into osmolarity data once this type of change is identified.This strategy can occur without intervention by medical professionals.That is, the chip processing can be initiated upon coupling to the baseunit and is not necessarily dependent on operating the lid of the baseunit or any other user intervention.

In any of the configurations described above, either the “smart chip”with processing circuitry on-chip (FIG. 7), or the electrode-onlyconfiguration with processing circuitry off-chip (FIG. 10), in apackaged chip (FIG. 7 and FIG. 10) or in a probe card (FIG. 11), thesample receiving chip can be disposed of after each use, so that thebase unit serves as a platform for interfacing with the disposablemeasurement chip. As noted, the base unit can also include relevantcontrol, communication, and display circuits (not shown), as well assoftware, or such features can be provided off-chip in the base unit orelsewhere. In this regard, the processing circuitry can be configured toautomatically provide sufficient power to the sample region electrodesto irreversibly oxidize them after a measurement cycle, such that theelectrodes are rendered inoperable for any subsequent measurement cycle.Upon inserted a used chip into the base unit, the user will be given anindication that the electrodes are inoperable. This helps preventinadvertent multiple use of a sample chip, which can lead to inaccurateosmolarity readings and potentially unsanitary conditions.

A secondary approach to ensure that a previously used chip is not placedback into the machine includes encoding serial numbers, or codesdirectly onto the chip. The base unit will store the used chip numbersin memory and cross-reference them against new chips placed in the baseconnector. If the base unit finds that the serial number of the usedchip is the same as an old chip, then the system will refuse to measureosmolarity until a new chip is inserted. It is important to ensure useof a new chip for each test because proteins adsorb and salt crystalsform on the electrodes after evaporation has run its course, whichcorrupt the integrity of the measuring electrodes.

In a further embodiment shown, in FIG. 12, the osmolarity of a samplefluid can be measured optically in an optical measurement system 1200 byusing optical indicators 1202 disposed on a measuring region 1212 of thechip substrate 1204. The optical indicators 1202 can comprise, forexample, nano-scale spheres, also called nanobeads, that are coated withchemicals whose luminescence varies with exposure to sample fluid ofvarying osmolarity, i.e. ionophores, or plasmon resonances. Thenanobeads 1202 can be deposited on the chip substrate 1204 on top of theelectrodes described above for the conductivity-measuring chips. Theelectrodes can be useful, e.g., for determining the volume of the samplefluid, as described above. However, other volume-measuring elements canbe used to determine the volume of the sample fluid. Preferably, but notnecessarily, the optical chip is produced with inert packaging such asdescribed above in connection with FIG. 7, including a chip opening holethrough which the collection device tip can be inserted. The samplefluid can then be expelled from the collection device and the samplefluid can come into contact with a predetermined, fixed number of thenanobeads per electrode site, which become immersed in the sample fluid.

When the nanobeads 1202 are illuminated with an optical energy source1210, such as a laser, the beads 1202 will fluoresce in accordance withthe osmolarity of the sample fluid 1206. The fluorescence can bedetected using a suitable optical detector light receiving device 1208,such as a conventional charge-coupled device (CCD) array, photodiode, orthe like. The resulting output signal of the light receiving array canindicate the osmolarity value of the sample fluid. It should be notedthat the nano-scale beads are sized such that an aliquot-sized fluidsample 1206, i.e., no more than 20 microliters of the fluid, willordinarily produce sufficient fluorescence to provide an output signalthat can be detected by the light receiving device 1208 and that canindicate osmolarity of the sample fluid. The amount of fluorescence canbe normalized by calculating how many nanobeads were activated by fluidand by measuring which electrode pairs were activated by the samplefluid. This normalization accounts for the sample volume and allows thevolume independence feature of the prior embodiment to be retained.

FIG. 13 is a flowchart describing an exemplary osmolarity measurementtechnique in accordance with one embodiment. First, a body fluid sample,such as a tear film, is collected in step 1300. The sample typically,e.g., contains less than one microliter. At step 1302, the collectedsample can be deposited on a sample region of a chip substrate. Theenergy properties of the sample can then be measured at step 1304. Themeasured energy properties can then be processed, at step 1306, todetermine the osmolarity of the sample. If the chip operates inaccordance with electrical conductivity measurement, then themeasurement processing at step 1306 can include the “electrodeoxidation” operation described above that renders the chip electrodesinoperable for any subsequent measuring cycles.

In the measurement process for a conductivity measuring system, asubstantially instantaneous shift is observed from the open circuitvoltage to a value that closely represents the state of the sample atthe time of collection, upon placement of a sample tear film on anelectrode array of the substrate. Subsequently, a drift in theconductivity of the sample will be reflected as a continual change inthe output.

The output of the measurement chip can be a time-varying voltage that istranslated into an osmolarity value. Thus, in a conductivity-basedsystem, more information than just the “electrical conductivity” of thesample can be obtained by measuring the frequency response over a widerange of input signals, which improves the end stage processing. Forexample, the calibration can be made over a multiple frequencies, e.g.,measure ratio of signals at 10, 20, 30, 40, 50, 100 Hz, etc. to make themeasurement process a relative calculation. This makes the chip-to-chipvoltage drift small. The standard method for macroscale electrode basedmeasurements, i.e., in a pH meter, or microcapillary technique, is torely upon known buffers to set up a linear calibration curve. Becausephotolithography is an extremely reproducible manufacturing technique,when coupled to a frequency sweep, calibration can be performed withoutoperator intervention.

As mentioned above, the processing of the energy properties can beperformed in a neural network configuration, where the seeminglydisparate measured data points obtained from the energy properties canbe used to provide more accurate osmolarity reading than from a singleenergy property measurement. For example, if only the electricalconductivity of the sample is measured, then the calibration curve canbe used to simply obtain the osmolarity value corresponding to theconductivity. This osmolarity value, however, generally will not be asaccurate as the output of the neural network.

The neural network can be designed to operate on a collection ofcalibration curves that reflects a substantially optimized transferfunction between the energy properties of the sample fluid and theosmolarity. Thus, in one embodiment, the neural network can beconfigured to construct a collection of calibration curves for allvariables of interest, such as voltage, evaporation rate, and volumechange. The neural network can also construct or receive as an input apriority list that assigns an importance factor to each variable toindicate the importance of the variable to the final outcome, or theosmolarity value. The neural network can be configured to construct thecalibration curves by training on examples of real data where the finaloutcome is known a priori. Accordingly, the neural network can betrained to predict the final outcome from the best possible combinationof variables. This neural network configuration that processes thevariables in an efficient combination can then be loaded into theprocessing unit residing, e.g., in the measurement chip 701 or the baseunit. Once trained, the neural network can be configured in softwareand/or hardware.

Although the embodiments described above for measuring osmolarityprovide substantial advantage over the conventional osmolarity measuringtechniques such as a freezing point depression technique, theembodiments described herein can be used to determine osmolarity of asample in accordance with the freezing point depression technique.Accordingly, the exemplary osmometry system 600 of FIG. 6 can be used toprovide an osmolarity value based on the freezing point depressiontechnique.

Such a freezing point depression system involves collecting anddepositing the sample fluid in a similar manner as in the steps 1300 and1302 illustrated in the flowchart in FIG. 13. As noted above, however,the osmometer of the osmometer system can include a cooling device, suchas a Peltier cooling device. In the embodiment of FIG. 7 describedabove, the Peltier device can be disposed on the socket 710 or the chip701 (see FIG. 7) to cool the sample. If desired, the Peltier coolingdevice can be used to cool the sample fluid to the freezing point of thesample fluid. A photo-lithographed metal junction, or p-n junction,known as a thermocouple, can be used to monitor the temperature ofaliquot-sized samples. The thermocouple can be configured to operate inparallel to the electrode array and Peltier cooling device, where thechip would be cooled below freezing so that the sample becomes a solid.Upon solidification, the electrical conductivity of the sample willdrastically change. Because the thermocouple is continually measuringthe temperature, the point at which the conductivity spikes can becorrelated to the depressed freezing point. Alternatively, the chip canbe supercooled immediately prior to sample introduction by the Peltierunit, and then by using the resistive heating inherent to theelectrodes, a current can be passed along the solid phase material. Uponmelting, the conductivity will again drastically change. In the secondmeasurement technique, it is likely that evaporation will be less of afactor. Thus, the embodiments described herein permit freezing pointdepression to be performed at significantly smaller volumes of samplefluid than previously possible.

With reference to FIG. 8 above, an embodiment of an integrated circuitcomprising a hole 720 was illustrated and described. As described, ahole 720 can be used to allow an aliquot volume of the sample fluid 702,e.g., tear fluid, to be deposited on the sample region 806. In theexample of FIG. 8, the hole is configured such that a collection device,e.g., a capillary 808, can be used to deposit the sample fluid 702 ontothe substrate 806. In other embodiments, however, hole 720 can comprisea channel configured to receive the sample fluid 702 through capillaryaction or negative pressure and cause it to be transferred to the sampleregion 806.

The ability to include such a channel can be important because it caneliminate a step in the process. For example, for the embodimentillustrated in FIG. 8, a two step process is required, wherein thesample fluid 702 is first collected and then deposited onto the samplesubstrate 806. Such a two step process can be sufficient for manyapplications; however, for some applications, e.g., involving tear film,such a two step process may not be sufficient. For example, in tear filmapplications, the amount of fluid can be very small. Accordingly, anyloses that occur during the two step process, e.g., due to evaporation,operator error, or the process itself, can cause erroneous results.Accordingly, limiting the chances for such losses can, in certainembodiments, greatly improve the efficiency and accuracy of the test,while simplifying the process.

In order to include such a microfluidic channel, the material selectionfor the packaging 707 can play an important role. This is because theability of the substrate to receive the sample fluid will dependsubstantially on the material chosen. Thus, the material chosen shouldallow for the rapid collection and transfer of the sample fluid, e.g.,tears. Accordingly, in certain embodiments, an appropriate glass orpolymeric material can be chosen to allow for the required rapidcollection of the associated sample fluid, while at the same timeallowing sufficient manufacturing tolerances so that the IC can bemanufactured affordably. For example, materials or surface treatmentswhich decrease the contact angle between the fluid, e.g., tears, and thesubstrate, preferably below 90°. A more detailed description of thematerials and material characteristics that can be used is presentedbelow.

Accordingly, a hole or a channel 720 can become a fluid, or tearcollection interface that can be used to receive a sample fluid andtransfer it to the sample region 806. It should be noted that theposition and geometry of the hole, or the channel 720 can vary in orderto optimize the collection and measurement of the sample fluid 702. Forexample, FIG. 14 is a diagram illustrating an IC 1400 comprising asample region 701, a transducer within the sample region 806, e.g.,electrodes, optical indicators, etc., with the upper strata of thesubstrate 707 encapsulating the sample region 701 and the lower strataof the substrate 804. In the example of FIG. 14, a channel 1402 isformed in the substrate 804 so as to receive tears, e.g., throughcapillary action. For example, a channel 1402 can be formed in substrate804 using various semiconductor manufacturing techniques. As describedabove, the dimensions and material chosen for the substrate 707 shouldbe selected to ensure rapid collection and transfer of the sample fluid702 to the sample region 806.

Semiconductor processing techniques can be used to form the channel 1402residing in the lower strata of the substrate 804. Again, the dimensionsand design of the channel 1402 should be selected taking into accountthe manufacturing tolerances of the semiconductor fabrication techniquesbeing used in order to optimize manufacturability. The design of thesubstrate should also promote tear collection. For instance, traditionalglass capillarie, promote tear collection, are often pulled to have acircular cross section, with a diameter of less than 300 micrometers(μm) with outer diameters of roughly 1 mm. Such a circular crosssection, however, may not be optimal, e.g., for tear collection.

Accordingly, the channel 1402 can be tapered at each end to improvecapillary action. This can also be achieved using a sandwichconstruction. Such a sandwich construction is shown in FIG. 16, whichillustrates a cross sectional view of the channel 1402 in accordancewith one embodiment. In the embodiment of FIG. 16, the sloped channel1402 can be a full width half max dimension of less than approximately200 μm with a smooth rise at the channel edges 1602 and 1604. Dependingon the embodiment, the rise can be sinusoidal, sigmoidal or Gaussian.These embodiments provide drastically shallower channel geometries nearthe lateral boundary of the channel than the center of the channel.Accordingly, these embodiments should display an increased capillaryforce at these boundaries, lowering the barrier to capillary action.This provides a substantial benefit over traditional glass capillaries,which feature cylindrical lumens.

The advantages of these geometries include a lower total volume perlength of the microchannel as compared to cylindrical channels, as wellas the ability to promote tear collection from the inferior fornix alsoknown as the lower tear lake, which is comprised of the thin meniscus offluid found at the interface between the lower lid andconjunctiva/cornea. These embodiments allow the surface tension of thetear fluid to bridge the opening of the channel when the tear collectioninterface is placed in the tear lake; fluid will “jump up” to cover thefront of the microchannel. Unlike a cylindrical construction where it isoften necessary to approach the tear lake perpendicular to the crosssection of the lumen, these embodiments promote rotation into the tearlake or resting the tear collection interface on the lower lid whichthen allows the surface tension to bridge the opening of the lumen.

In other embodiments, the channel 1402 can comprise a triangular crosssection, a rounded triangle cross section, a half circle cross section,etc. In fact, the channel 1402 can comprise any geometry that promotesfluid collection.

Other limitations of traditional capillaries are also overcome throughthe substrate configurations noted herein. For instance, the needle-likeappearance of a glass capillary is suboptimal for patient interaction.In accordance with the embodiments described herein, the substrate canbe made rounded or more blunt edged to be more inviting to the patient,and can be made of softer materials, i.e., polymer, to eliminate thechance of injury to the corneal surface. Furthermore, the edge of thesubstrate can be configured to be very thin near the opening of thechannel to promote entrance into the tear lake. For example, in oneembodiment the sample receiving chip can be placed parallel to the lowerlid and then rotated upwards such that the tip of the substrate touchesthe tear lake. By fashioning the substrate such that the upper stratacovering the channel is minimized in vertical extent, preferably lessthan approximately 100 μm, the substrate can be rotated into the tearlake and the lumen of the channel can be completely covered with tear,even if the entirety of the substrate is not wetted. The lower strata ofthe substrate can also be configured for mechanical stability.

These techniques are not possible with traditional glass capillaries,which are radially symmetric and lose mechanical stability as the lumenof the capillary approaches 100 μm. In this case, collection of smallnanoliter volumes presents undue risk to the patient as a properly timedblink could break off the tip of the glass capillary and introduceshards of glass into the patient's eye. Asymmetrically configuring thestrata of the substrate provides both the ability to use rotational tearcollection as well as superior mechanical stability, capillary action,and patient interaction as compared to traditional tear collectionmethodologies. These embodiments are particularly useful when thepatient lacks a substantial volume of tear on their ocular surface.

The substrate can also be configured to promote capillary action byhaving a curved shape having an apex or rounded peak so that thesubstrate can be moved proximate the eye surface, to pull in tear fluidvia capillary action. The curved shape can be configured to minimize thesubstrate area that comes close to the eye, thereby minimizing anycontact with the eye and making it easier for the clinician to get closeto the eye and collect the fluid. The apex of the curve can include afeature to promote capillary action and receive the fluid. For example,FIG. 17B shows that a channel 1402 in the substrate 1704 extends to theedge of the shape, at the apex. Placing the edge of the substrate 1704proximate the eye surface allows tear fluid to enter the channel 1402,utilizing a capillary action.

It will be understood that conventional semiconductor manufacturingtechniques can handle such dimensions and features. Thus, the channel1402 can be patterned and formed, e.g., using an excimer laser, Nd-YAGlaser, or photolithography. The material chosen should then be amenableto the process being used.

It should be noted that the upper strata of the substrate 707 can beformed such that channel 1402 extends to the edge of the substrate. Inthis way, the IC 1400 can act as the receiving substrate for collectingsample fluid 702. Thus, rather than using a separate collection device,such as a glass capillary to collect the fluid, the IC 1400 can be usedfor collection and measurement. Thus, the IC 1400 can comprise asubstrate that promotes tear collection. The IC 1400 can then beinterfaced with a processing device, such as device 606, or in otherembodiments, removed from the collection device and interfaced with aprocessing device.

FIGS. 17A-17C are diagrams illustrating example embodiments of a samplereceiving chip 1700 configured in accordance with one embodiment.Integrated circuit 1700 can comprise a substrate 1704, which can includea sample region 1706, which is shown in finer detail in FIG. 17C. Thesubstrate 1704 can also include a channel 1402 and electrodes 1710. Theupper strata of the substrate 1702 can be placed over the lower strataof the substrate 1704 as illustrated in FIG. 17A. As can also be seen inFIG. 17B, the channel 1402 can extend to the edge of the device 1700 sothat the substrate 1704 can receive an aliquot volume of tear andtransfer the fluid to the sample region 1706 for measurement. Note thatthe substrate 1704 is shaped to promote capillary action from the tearlake as described above. The curved edge of the substrate 1704 with thechannel 1402 placed perpendicular to the tangent of the curved edgepromotes capillary action within the tear lake with minimal risk to thepatient. The substrate shape includes the curved edge of the substrate1704, the appropriate thickness of the strata 1702 and 1704, and thecross section of the substrate channel 1402. The substrate shape canalso comprise a short, blunt end with channel 1402 perpendicular to theblunt end, and then a linearly receding substrate to form arectangularly or triangularly receding shape to the substrate 1704.

The substrate 1704 can also be shaped to promote easy placement near theeye surface, such that the sample receiving chip can be rotated, dipped,pressed, or linearly translated into the tear lake while exposing thechannel edge of the substrate to the tear lake. The substrate can alsobe shaped such that it is gently angled to allow the channel to protrudeslightly, which allows a thinner extent that makes contact with the tearlake. Since the channel 1402 extends from the sample region 1706 to theedge of the substrate at the rounded edge, the shape of the substratetherefore promotes wicking through capillary action from the edge to thesample region.

FIG. 17B is a diagram illustrating a blown up view of area 1706 in FIG.17A. As can be seen, electrodes 1710 can be formed over substrate 1704and in contact with channel 1402 in the sample region. FIG. 17B alsomore clearly illustrates that channel 1402 extends to the edge ofsubstrate 1402, and therefore the edge of device 1700.

It should be noted that channel 1402 does not necessarily need to havethe shape and geometry illustrated in FIGS. 17A and 17B. As mentionedabove, the channel 1402 can comprise any one of various cross sectiondimensions and in general, the channel 1402 can comprise any geometrythat promotes fluid collection. Moreover, the channel 1402 can actuallycomprise any modification to the surface of substrate 1702 that performsthe functions of fluid collection.

In the example embodiment of FIGS. 17A and 17B, the upper strata of thesubstrate 1704 can be made from a polyester film and attached via ahydrophilic adhesive applied to the bottom side of the substrate 1704.The substrate 1704 can be formed from a polycarbonate material or othermaterial compatible with semiconductor fabrication techniques. Thesubstrate materials are preferentially hydrophilic, although a sandwichconstruction (see FIG. 16), where a hydrophilic layer seals a morehydrophobic channel, or hydrophobic sealant of a hydrophilic channel,can also be made to wick tears; glass on polyimide, for instance. Theclass of materials that are preferable for the substrate include glass,hydrophilic polymers, silicon, di- and triblock copolymers with amides,amines, sulfates, phosphates or other charged groups. For instance,polyether block amides (PEBA), block-copolyether-esters (PEE),polylactic acid (PLA), polyurethanes (PU) including aliphatic andthermoplastic polyurethanes, polyglycolic acid (PGA) and otherpolyesters (PE), polycaprolactone (PCL), polyethersulfones (PES),polycarbonate (PC) or any other combination of hydrophilic copolymerswhich demonstrate proper manufacturing stability and contact angle whichpromote tear collection.

Other means of constructing a heterogeneous substrate include astratified stack of materials that promote wetting at the tear filminterface as well as hydrophilicity throughout the extent of the samplereceiving region of the substrate 1402. For instance, in one embodiment,a hydrophilic pressure sensitive adhesive (PSA) is used to seal apolycarbonate channel to a glass cover slip, such that the strata(glass, PSA, polycarbonate) decrease in hydrophilicity, yet when placedin the tear lake, the tear fluid readily wets across the lumen ofchannel 1402. In such an embodiment, the upper strata of the substrate1702 can be made of any of the aforementioned materials, which reducesurface tension and promote wetting when placed in contact with the tearfilm. Similar configurations are possible when making the lower strataof the substrate 1704 hydrophilic through intrinsic material propertiesor surface treatments.

In other embodiments, a polycarbonate substrate adhered to a hydrophilicPSA comprised of, e.g., 25 μm polyester-based hydrophilic adhesive witha, e.g., 100 μm polyethylene terephthalate (PET) backing can be used. Tocomplete the stack, a hydrophobic adhesive can be applied around theoutside of the substrate 1714 to eliminate the flow of tears around theexterior of the substrate 1704. Such an embodiment is pictured in FIG.18, with the substrate 1704, the hydrophilic PSA 1702, and thehydrophobic adhesive 1800 pictured. The hydrophobic adhesive can becomprised of, e.g., beeswax, epoxy resins, or UV curable resins such asurethane (meth) acrylate, and the like. The absence of adhesive 1800 canallow tears to flow around the exterior of the substrate 1704 and shortout the electrodes at the back of PSA 1702.

In another embodiment, the tear collection interface can use thesigmoidal, sinusoidal, or semicircular channel from the PSA backing andhydrophilic PSA adhesive, with the electrodes residing on a flatpolycarbonate substrate.

Another embodiment of hydrophilic strata uses identical material on theupper and lower strata but includes a hydrophilic layer in the middle,in direct contact with the channel lumen 1402. This can be a lessexpensive construction. Amphiphilic polymer constructions can also beused, where hydrophobic side chains are used to bond strata together,while exposing hydrophilic side chains to the channel interior.

Modifications to one or more of the material layers can also promotetear collection, such as plasma etching, with nitrogen, oxygen, argon orother gaseous plasmas, acid treatment, exterior coating, increases insurface roughness on the micro- or nanoscale, or comparable methods thatreduce contact angle. For example, polyelectrolyte coatings comprisingpolyethyleneimine, polyaminoalkyl methacrylate, polyvinylpyridine,polylysine, polyacrylic acid, polymethacrylic acid, polysulfonic acid,polyvinyl sulfate, polyacrylamido-2-methyl-1-propanesulfonic acid, andpolystyrene sulfonic acid, or other coatings or resin additives known toincrease charge density at the interface. In general, any material,e.g., polymer, resin, glass, etc., can be used for the substrate 1702that can promote capillary action at the edge of a the sample receivingchip.

FIG. 15 is a diagram illustrating an example of the collection device1500 comprising, e.g., a sample receiving chip 1700 in accordance withone embodiment. Device 1500 can, for example, be sized and shapedsomewhat like an pen and can comprise a base portion 1502 and a tipportion 1504 configured to house the sample receiving chip 1700. The tipportion 1504 can be configured so that it can be placed in contact withthe sample fluid allowing channel 1402 to collect an aliquot volume ofthe sample fluid for testing. Tip portion 1504 can be configured so thatit can then be removed and interfaced with a processing unit 606,thereby interfacing device 1700 with processing unit 606 so that theosmolarity of the sample fluid can be measured as described above. Thus,collection device 1500 can include a mechanism (not shown) fordecoupling, or ejecting tip portion 1504. Collection device 1500 canalso be a blunt ended, flat device that seems less needle like to thepatient and uses a hinge mechanism to receive device 1700.

In certain embodiments, tip portion 1504 and/or device 1700 can then bedisposed of and base portion 1502 can be reused with another tip portion1504 and/or device 1700. Methods such as those described above can thenbe used to ensure that a previously used device 1700 is not reused.

Further, an informational signal 1508 can be integrated withincollection device 1500 and configured to indicate whether the substrateis properly connected and whether enough sample fluid has beencollected. For example, fluid filled electrodes, e.g., the outermostelectrodes shown in FIG. 17A, i.e., closest to the channel opening, andclosest to the vent hole, can provide a convenient transducer within thesubstrate. A 2-point impedance measurement across these electrodes candistinguish between an open circuit device, and an attached substratewith an empty channel, with typical impedance values changing fromaround 5 MOhm to around 1 MOhm upon connection of the substrate to thedevice. Tear collection reduces the impedance between the fluid fillelectrodes to generally below 100 kOhm at 100 kHz, providing two clearthresholds for hardware to provide user feedback.

Indicator 1508 can also include, or be coupled with an auditoryindicator to indicate whether enough sample fluid has been collected.For example, a Light Emitting Diode (LED) or other indicator can beactivated when enough sample fluid is present. A beep or other tone inconjunction with the visible feedback can be used as parallel indicationof filling the channel. Alternatively, one indicator, such as a red LED,can be included to indicate that not enough sample fluid is present, anda second indicator, such as a green LED can be used to indicate whenenough sample fluid is present. Thus, for example, the red LED can beactive until enough sample fluid is present at which point the red LEDis turned off and the green LED is activated.

In other embodiments, audible indicators can be used. In still otherembodiments, displays such as LED or Liquid Crystal Displays (LCDs) canbe used to convey the sample fluid status.

The embodiments described above are generally related to systems andmethods for detecting, or determining osmolarity for a fluid sample;however, it will be appreciated that the systems and methods describedherein are not limited to the detection/determination of osmolarity.Rather, the systems and methods described herein can be employed todetect other parameters associated with a sample fluid. For example, inthe embodiments described below, the systems and methods describedherein can be used to detect any analyte of interest contained in thefluid sample. For example, the systems and methods described herein canbe used to detect analytes such as proteins, peptides, andoligonucleotides. More specifically, the systems and methods describedherein can be used to detect, or measure any immunoglobulin, such asImmunoglobulin E (IgE), which can be useful for testing for allergies,Immunoglobulin M (IgM), Immunoglobulin A (IgA), Immunoglobulin M (IgM),etc. The systems and methods described herein can also be used to detectany cytokine, protein, or mucin, such as TGF-Beta, TNF-alpha,Interleukin 1-A, MUC5, or PRG4.

More broadly, the systems and methods described herein can be used todetect or measure various biomarkers in the sample fluid. For example,the systems and methods described herein can be used to detectbiomarkers in tears, such as osmolarity, IgE, lactoferrin, cytokines,etc. For example, in certain embodiments, electrical signals produced byelectrodes in sample region 806 can be used to detect analytes such asproteins. In other embodiments, however, optical detection methods canbe used to detect analytes of interest. In general, any of varioustransduction techniques can be used to detect or measure an analyte ofinterest. For example, electrochemical, optoentropic, optomechanical,fluorescent, chemiluminescent, chromataographic, surface plasmonresonant (SPR) transduction methods can be used to detect analytes in afluid sample incident on sample region 806. In other embodiments,nanobeads can be used to detect analyte of interest in the sample fluid.For example, the nanobeads can be coated with a chemical that changesfluorescence based on the amount of the target analyte present in thesample fluid. In other embodiments, the nanobeads can be coated with abiological substance that binds to the analyte of interest. Light canthen be used to illuminate the beads and detect the presence of theanalyte, e.g., using SPR or by detecting fluorescence, luminescence, orother changes in the energy properties of the sample region. Othertransduction mechanisms such as electrochemical includingpotentiometric, amperometric, capacitance, and impedance spectroscopy,cyclic voltammery, pulse voltammery, etc., transduction methods can beused in conjunction with the electrodes within the sample region. Enzymemodified electrochemical redox reactions, such as horseradish peroxidaselabels, gold nanoparticle labels, and other electrochemically activelabels can be used within the transduction mechanism. Furtherembodiments can include measuring changes in potentiometric conductivepolymers, such as polypyrrole, after exposure to tear fluid.

Conductive polymers and the other transduction systems described hereincan be incorporated directly into the sample region of the substrate inorder to mitigate the effects of evaporation on an open system.

In addition to physically locate the transduction system within thecollection interface, two other methods of mitigating evaporation duringmeasurement of analytes of interest include a sealing cap for the tearcollection interface, as well as the use of software to normalize theanalyte of interest against the osmolarity of the sample. The cap couldbe comprised of an interference fitted plastic, or gasketed seal, muchlike a normal pen cap, which slides over both the vent hole of thesubstrate and channel opening 1402. The cap design could allow for avery small displacement of air, as the movement of fluid within thechannel is undesired. For example, vent holes that are carved along theoutside of the pen cap could terminate just prior to sealing such thatsufficient mechanical stability is achieved while minimizing the airdisplacement.

Osmolarity normalization can be calculated to compensate for theintrinsic evaporation while the tears are within the patient's tearfilm, as well as for osmolarity changes during residence within thechannel. During measurement of analytes of interest, biochemical assaysoften require incubation times that can be significantly longer than thetimes needed to measure the impedance of the tear fluid. FIG. 19demonstrates a typical change in osmolarity over time within a receivingsubstrate as measured by a four-point impedance method. The initialtransient, within the first 10 seconds, sees the impedance increase as aresult of the equilibration (slowing) of the tear fluid being pulledinto the capillary channel. Often, a small volume of residual tearremains outside of the substrate immediately following tear collection.As these tears are exposed to the environment with a large surface area,these tears are of higher osmolarity than the tears that originallypopulated the channel. Continual capillary action draws this higherconcentration fluid into the channel and mixes the fluids, graduallydecreasing the impedance of the fluid as the concentration changes. Asthe residual tear source is lost, the flow of the fluid begins to slowagain, e.g., about 140 seconds in, increasing the impedance. If the venthole contains a reservoir of fluid, it is typically hypoosmolar at thebase of the column, the fluid unexposed to the air. Once the vent startssourcing the fluid back into the channel, the hypoosmolar fluid lowersthe concentration of the fluid and increases the impedance. After allsources have been emptied, the impedance drops in a near-linear fashion,indicative of steady evaporation.

FIG. 20 exhibits these dynamics across three different tear collectioninterface geometries. The smaller the channel, the larger the gain ofthe dynamics. As can be seen, a 100 μm wide, by 75 μm deep, by 2.5 mmlong sinusoidal channel constructed from polyester PSA thermally bondedto polycarbonate, has the greatest percent change in osmolarity duringincubation, with about a 34% change over 150 seconds. A 300 μm wide, by75 μm deep, by 5 mm long channel sees only a few percent change over thetime of incubation.

The transduction of analytes of interest can be normalized against thesedynamics. For instance, an instantaneous potentiometric measurement canbe normalized against the ratio of the initial steady state value, e.g.,around 10 seconds, vs. the instantaneous impedance at the time ofmeasurement as one of the methods of normalization. Integralamperometric methods can be normalized against the average of thedisplacement of the impedance curve. In general, many normalizations canbe made to adjust the reported level of analyte of interest in order toimprove the standard of care.

The systems and methods described herein have been described above interms of exemplary embodiments so that an understanding of the presentinvention can be conveyed. Any embodiment described herein as“exemplary” is not necessarily to be construed as preferred oradvantageous over other embodiments. Moreover, there are manyconfigurations for the systems and associated components notspecifically described herein but with which the present invention isapplicable. The systems and methods described herein should thereforenot be seen as limited to the particular embodiments described herein,but rather, it should be understood that the present invention has wideapplicability. For example, in addition to tear film and osmolarity, thesystems and methods described herein can be used for any fluid, e.g.,serum, and to detect a variety of parameters including osmolarity andthe presence, or amount of an analyte of interest. All modifications,variations, or equivalent arrangements and implementations that arewithin the scope of the attached claims should therefore be consideredwithin the scope of the invention.

1. A sample receiving chip, comprising: (a) a substrate that receives analiquot volume of a sample fluid, wherein the substrate is operativelyshaped to receive the aliquot volume of sample fluid through capillaryaction; and (b) a sample fluid region of the substrate, sized such thatthe volume of the sample fluid is sufficient to operatively cover aportion of the sample region, whereupon energy properties of the samplefluid can be transduced to produce a sample fluid reading; wherein thesample fluid reading indicates (i) the osmolarity of the sample, and(ii) the presence or amount of an analyte of interest in the samplefluid.
 2. The sample receiving chip of claim 1, wherein the analyte ofinterest is a protein, peptide, or oligonucleotide.
 3. The samplereceiving chip of claim 1, wherein the analyte of interest is animmunoglobulin, a cytokine, a mucin, or a combination thereof.
 4. Thesample receiving chip of claim 1, wherein the analyte of interest is anIgM, IgA, IgE, or combination thereof.
 5. The sample receiving chip ofclaim 1, wherein the analyte of interest is TGF-β, TNF-α,interleukin-1-A, MUC5, PRG4, lactoferrin, or a combination thereof.
 6. Asample receiving chip, comprising: (a) a substrate that receives analiquot volume of a sample fluid, wherein the substrate is operativelyshaped to receive the aliquot volume of sample fluid through capillaryaction; and (b) a sample fluid region of the substrate, sized such thatthe volume of the sample fluid is sufficient to operatively cover aportion of the sample region, whereupon energy properties of the samplefluid can be transduced to produce a sample fluid reading; wherein thesample fluid reading indicates the presence or amount of an analyte ofinterest in the sample fluid.
 7. The sample receiving chip of claim 6,wherein the sample fluid is a bodily fluid.
 8. The sample receiving chipof claim 6, wherein the sample fluid is tear film.
 9. The samplereceiving chip of claim 6, wherein the analyte of interest is a protein,peptide, or oligonucleotide.
 10. The sample receiving chip of claim 6,wherein the analyte of interest is and immunoglobulin, a cytokine, amucin, or a combination thereof.
 11. The sample receiving chip of claim6, wherein the analyte of interest is an IgM, IgA, IgE, or combinationthereof.
 12. The sample receiving chip of claim 6, wherein the analyteof interest is TGF-β, TNF-α, interleukin-1-A, MUC5, PRG4, lactoferrin,or a combination thereof.
 13. A sample receiving chip, comprising: (a) asubstrate that receives an aliquot volume of a sample fluid, wherein thesubstrate is operatively shaped to receive the aliquot volume of samplefluid through capillary action; and (b) a sample fluid region of thesubstrate, sized such that the volume of the sample fluid is sufficientto operatively cover a portion of the sample region, whereupon energyproperties of the sample fluid can be transduced to produce a samplefluid reading; wherein the sample region comprises a plurality ofoptical indicators, and wherein the optical indications comprise aplurality of nano-scale spheres that luminesce in the presence of ananalyte of interest.
 14. The sample receiving chip of claim 13, whereinthe sample fluid is a bodily fluid.
 15. The sample receiving chip ofclaim 13, wherein the sample fluid is tear film.
 16. The samplereceiving chip of claim 13, wherein the analyte of interest is aprotein, peptide, or oligonucleotide.
 17. The sample receiving chip ofclaim 13, wherein the analyte of interest is an immunoglobulin, acytokine, a mucin, or a combination thereof.
 18. The sample receivingchip of claim 13, wherein the analyte of interest is an IgM, IgA, IgE,or combination thereof.
 19. The sample receiving chip of claim 13,wherein the analyte of interest is TGF-β, TNF-α, interleukin-1-A, MUC5,PRG4, lactoferrin, or a combination thereof.